Affiliations
Department of Biology, University of
Washington, Seattle, Washington, United States of America,
Graduate Program in Molecular and Cellular
Biology, University of Washington, Seattle, Washington, United States of
America

Affiliations
Department of Biology, University of
Washington, Seattle, Washington, United States of America,
Graduate Program in Biology, University of
Washington, Seattle, Washington, United States of America

Figures

Abstract

The pigment cells of vertebrates serve a variety of functions and generate a
stunning variety of patterns. These cells are also implicated in human
pathologies including melanoma. Whereas the events of pigment cell development
have been studied extensively in the embryo, much less is known about
morphogenesis and differentiation of these cells during post-embryonic stages.
Previous studies of zebrafish revealed genetically distinct populations of
embryonic and adult melanophores, the ectotherm homologue of amniote
melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging,
mutational analyses, and transgenesis to identify peripheral nerves as a niche
for precursors to adult melanophores that subsequently migrate to the skin to
form the adult pigment pattern. We further identify genetic requirements for
establishing, maintaining, and recruiting precursors to the adult melanophore
lineage and demonstrate novel compensatory behaviors during pattern regulation
in mutant backgrounds. Finally, we show that distinct populations of latent
precursors having differential regenerative capabilities persist into the adult.
These findings provide a foundation for future studies of post-embryonic pigment
cell precursors in development, evolution, and neoplasia.

Author Summary

Understanding the biology of post-embryonic stem and progenitor cells is of both
basic and translational importance. To identify mechanisms by which stem and
progenitor cells are established, maintained, and recruited to particular fates,
we are using the zebrafish adult pigment pattern. Previous work showed that
embryonic and adult pigment cells have different genetic requirements, but
little is known about the molecular or proliferative phenotypes of precursors to
adult pigment cells or where these precursors reside during post-embryonic
development. We show here that post-embryonic pigment cell precursors are
associated with peripheral nerves and that these cells migrate to the skin
during the larval-to-adult transformation when the adult pigment pattern forms.
We also define morphogenetic and differentiative roles for several genes in
promoting these events. Finally, we demonstrate that latent precursor pools
persist into the adult and that different pools have different capacities for
supplying new pigment cells in the context of pattern regeneration. Our study
sets the stage for future analyses to identify additional common and essential
features of pigment stem cell biology.

Funding: This research supported by National Institute of General Medical Sciences
(http://www.nigms.nih.gov/), National Institutes of Health grant
R01 GM062182 to DMP. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

A fundamental challenge for modern developmental biology is to determine how
populations of stem and progenitor cells are established, maintained, and recruited
to differentiate at particular times and places during post-embryonic development
and in the adult organism. The significance of the problem cannot be overstated. Not
only are these cells essential for normal development and homeostasis, but
understanding their biology has profound translational importance. If we seek to
evoke regenerative responses in a clinical content, then post-embryonic stem and
progenitor populations may well supply the cells for doing so [1]–[3]. If we hope to delay natural tissue
senescence, it is the life cycle of these cells that may need to be manipulated
[4]–[7]. And if we
aim to control malignancy, these cells or their transformed progeny will often be
our targets of choice [8]–[10].

Pigment cells are of great utility for understanding the biology of post-embryonic
stem and progenitor cells. Pigment cells are a classic and enduring system for
studying morphogenesis and differentiation, and a century of effort has provided a
firm understanding of many aspects of pigment cell development in the embryo [11]–[14]. These cells
arise from neural crest cells, which migrate from the dorsal neural tube and
contribute not only to pigment cells, but also glia and neurons of the peripheral
nervous system, bone and cartilage of the craniofacial skeleton, and more. Despite
the long-standing interest in these embryonic events, it is now clear that pigment
cells of adults derive in large part from post-embryonic stem cells that are
themselves of neural crest origin [15]–[18]. We know some of the mechanisms
that underlie post-embryonic precursor development yet many outstanding questions
remain. Foremost among these concern the genes and cellular behaviors by which
pigment stem or progenitor cells are established during early development and
subsequently maintained, whether there exist distinct subpopulations of such cells
with different genetic requirements and potentials, and how these cells are
recruited during normal development and homeostasis.

Answers to these questions will provide insights into the basic biology of the adult
pigment cell lineage, and can inform our understanding of post-embryonic neural
crest derivatives as well as stem and progenitor cells more generally. These answers
are also of enormous biomedical significance, as the skin pigment cell of mammals,
the melanocyte, is associated with a variety of human pathologies [19] and
transformed cells of this lineage give rise to melanoma, one of the most common
cancers [20], [21] and also one of
the most deadly [21]–[25]. Poor outcomes reflect the inefficacy of non-surgical
treatments and the highly invasive character of melanoma cells [26]–[29]. This invasiveness results in
part from neural crest and melanocyte-specific factors that are already expressed by
untransformed precursors, as well as lineage-specific factors that are re-expressed
upon transformation [30]. Better understanding the genetic requirements and
dynamics of melanocyte development and homeostasis can thus provide insights into
the behaviors of transformed cells, and may suggest novel strategies for clinical
intervention.

In recent years, the zebrafish has proven to be a tractable system for elucidating
features of pigment cell development in the embryo and during the larval-to-adult
transformation, a period of post-embryonic development analogous to later
organogenesis, fetal and neonatal development of mammals [31]–[33]. In the embryo, neural crest
cells differentiate into embryonic/early larval melanophores, the zebrafish
homologue of mammalian melanocytes. Melanophores and melanocytes depend on many of
the same genes and pathways [12], [14], [34], and melanomas with characteristics equivalent to those
of human melanomas can be induced in zebrafish [18], [34], [35].

The development of zebrafish adult pigmentation involves a “pigment pattern
metamorphosis” in which an embryonic/early larval pigment pattern is
transformed into that of the adult [12], [33], [36]–[38]. Whereas the embryonic/early larval pigment cells and
pattern develop by 3.6 SSL (standardized standard length [33]; about 4 days
post-fertilization), new “metamorphic” melanophores begin to
differentiate scattered over the flank by 5.9 SSL and ultimately migrate to form the
adult stripes. Simultaneously, additional metamorphic melanophores appear already at
sites of stripe formation, and many embryonic/early larval melanophores are lost.
These events culminate in a juvenile pigment pattern by 11.0 SSL (4–5 weeks
post-fertilization), consisting of two melanophore stripes bounding a lighter
“interstripe”. Melanophores comprising these stripes reside in the
“hypodermis” between the epidermis and the myotomes [39], [40]. Other adult
melanophores are found in the epidermis, the dorsal scales, and the fins. Two
additional classes of pigment cells also develop: yellow–orange xanthophores,
which populate the interstripe and are required for organizing melanophores into
stripes [38],
[41], [42]; and
iridescent iridophores, which are initially limited to the interstripe but later
occupy melanophore stripes as well [33]. During later adult development, additional stripes and
interstripes are added as the fish grows.

Mutants with pigment pattern defects limited to post-embryonic stages have suggested
a model in which embryonic/early larval melanophores develop directly from the
neural crest, whereas metamorphic melanophores develop from latent stem cells of
presumptive neural crest origin. For example, picasso and
puma mutants have normal embryonic/early larval melanophores,
but profound deficiencies in their complements of metamorphic melanophores.
picasso encodes the neuregulin receptor erbb3b, which acts both
autonomously and non-autonomously to the metamorphic melanophore lineage.
Pharmacological inhibition of ErbB signaling further revealed that
erbb3b activity is required during neural crest migration for
the later development of metamorphic melanophores, suggesting this locus is
essential for establishing a pool of precursors that will differentiate only later
during the larval-to-adult transformation [43]. By contrast,
puma encodes tubulin alpha 8-like 3a (tuba8l3a) and acts
autonomously to the metamorphic melanophore lineage. The temperature sensitivity of
this allele allowed the identification of a critical period during pigment pattern
metamorphosis, suggesting a role in maintaining or expanding a population of latent
precursors, or recruiting these cells as melanophores [36], [44], [45].

To date it has not been known where latent precursors to metamorphic melanophores
reside, how erbb3b, tuba8l3a or other loci promote
the normal morphogenesis and differentiation of these cells and their progeny, or
whether pigment cell precursors have indefinite or more limited re-population
potential. Here we investigate these issues using molecular marker analyses,
transgenesis, vital labeling, and time-lapse imaging in wild-type and mutant
backgrounds. We show that during post-embryonic development, proliferative pigment
cell precursors are associated with peripheral nerves and ganglia, and migrate to
the hypodermis during pigment pattern metamorphosis where they differentiate as
melanophores and iridophores. Nerve-associated pigment cell precursors are missing
or reduced in ErbB-deficient and tuba8l3a mutant backgrounds. By
contrast, these precursors persist in other mutants having less severe metamorphic
melanophore deficiencies, though their subsequent development is marked both by
defects, and partial regulation, of morphogenesis and differentiation. Finally, we
show that latent precursors persist into the adult but that different precursor
pools have different regenerative potentials. These findings provide a critical
context for understanding the cellular bases of adult melanophore development, the
mechanistic underpinnings of mutant phenotypes, and the roles for latent precursors
in adult homeostasis, regeneration, and neoplasia.

Results

A melanogenic, erbb3b-dependent population of
extra-hypodermal progenitor cells

To identify tissues that might harbor latent precursors to adult melanophores, we
examined post-embryonic zebrafish for transcripts expressed by embryonic neural
crest cells, reasoning that some of the cells expressing such markers might
comprise a population of undifferentiated melanophore precursors. We examined
foxd3 and sox10, which are expressed by
early neural crest cells, subpopulations of neural crest-derived glia, and some
other cell types [46]–[49], as well as
crestin, which is known only to be expressed by neural
crest cells and their derivatives [50]. Cells expressing these loci were present in the
hypodermis where the adult pigment pattern forms, but also in the myotomes,
adjacent to the spinal cord, and at the bases of the fins (Figure 1A–1G), raising the possibility
of both hypodermal and “extra-hypodermal” precursors for metamorphic
melanophores.

Shown are in situ hybridizations performed on transverse sections of
7–9 SSL larvae. (A) foxd3 transcript was detected
in dorsal root ganglia (arrow) and scattered cells (e.g., arrowheads)
within the myotome (m) and near the epidermis (e). (B)
sox10 expression by cells adjacent to the neural
tube (arrow) and within the myotome (arrowhead). (C)
foxd3+ cells (e.g., arrowheads) at the base of
the dorsal fin (f). (D) foxd3+ cells within the
myotomes and near the epidermis. (E–G)
foxd3+, sox10+, and
crestin+ cells within the myotomes.
(H–J) Cells expressed mitfa (arrowheads), within
the horizontal myoseptum (H), at the surface of the myotome (I), and at
the base of anal fin (J) (see text for details).

If extra-hypodermal, post-embryonic cells expressing genes typical of early
neural crest cells contribute to metamorphic melanophores, we hypothesized that
some of these cells should differentiate if supplied with appropriate trophic
support. To test this idea, we used a heat-shock inducible transgenic line,
Tg(hsp70::kitla)wp.r.t2, to misexpress the
melanogenic factor kit ligand-a (kitla) [51] throughout the larva
(Figure 2A, 2B). These
larvae developed ectopic melanophores deep within the myotomes, which were never
found in identically treated siblings lacking the transgene (Figure 2C–2G).

Previously we showed that erbb3b is essential for establishing
precursors to metamorphic melanophores [43]. Accordingly, if
extra-hypodermal kitla-responsive melanogenic cells and metamorphic melanophores
arise from a common precursor pool, then ectopic melanophores should fail to
develop in erbb3b mutants misexpressing kitla. Indeed,
erbb3b; Tg(hsp70:kitla) larvae exhibited ∼30-fold fewer
ectopic melanophores than wild-type Tg(hsp70::kitla) larvae
(Figure 2H). A similar
outcome was observed when wild-type larvae were treated with the ErbB inhibitor,
AG1478, during the embryonic critical period for erbb3b
activity in establishing metamorphic melanophore precursors [43]. Together,
these results indicated that some extra-hypodermal cells are competent to
differentiate as melanophores in the wild-type, but that most of these cells are
missing when erbb3b activity is lost.

Extra-hypodermal melanophore precursors are nerve-associated, proliferative,
and specified for the melanophore lineage during the larval-to-adult
transformation

Our identification of extra-hypodermal cells expressing genes typical of early
neural crest and glial cells, as well as kitla-responsive melanogenic cells
within the myotomes, led us to ask whether any of these cells embark upon a
melanophore differentiation program during normal post-embryonic development.
Since precursors to metamorphic melanophores require erbb3b, we
further predicted that any candidate extra-hypodermal precursors of these cells
should be missing in larvae deficient for erbb3b activity.

In the wild-type, we found extra-hypodermal cells expressing
mitfa, encoding a master regulator of melanophore fate
specification (Figure
1H–1J) [52], [53]. Cells expressing mitfa in
extra-hypodermal locations typically did so at lower levels than cells within
the hypodermis and were at the limit of detection given the sensitivity of in
situ hybridization at post-embryonic stages. However, we also identified
extra-hypodermal cells expressing GFP driven by the proximal
mitfa promoter in the transgenic line,
Tg(mitfa::GFP)w47, which faithfully
recapitulates mitfa expression in the melanophore lineage and
in bipotent precursors to melanophores and iridophores in the embryo [54], [55] (Figure 3A; Videos S1,
S2).
In contrast to the wild-type, mitfa::GFP+ cells were largely absent from
both erbb3b mutants and wild-type larvae treated with AG1478
during the embryonic erbb3b critical period (Figure 3B and see below; Video S3).
In neither genetic background could we detect extra-hypodermal cells expressing
transcript for dct, encoding a melanin synthesis enzyme.

All images are from early metamorphic (6.2–8.0 SSL) wild-type
larvae except for B, L, and M, from erbb3b mutant
larvae. (A–C) Transverse confocal projections (collapsing ∼1.5
mm of trunk along the anterior-posterior axis) showing GFP+ cells
in living larvae (left side of each larva is shown). Images correspond
to Videos S1, S2, S3.
(A) mitfa::GFP+ cells in a wild-type fish occur in the hypodermis,
between the epidermis (e) and the myotome (m), within the the myotome
itself (arrow), and above the spinal cord (sc). Arrowhead, location of
the horizontal myoseptum. (B) In erbb3b mutants, most
mitfa::GFP+ cells were missing. This image is intentionally
overexposed compared to A, revealing faint reflected fluorescence from
iridescent iridophores in the hypodermis (arrowhead), which are present
in wild-type larvae as well. (C) sox10::GFP+ cells were found in
extra-hypodermal locations of wild-type
Tg(−4.9sox10:egfp)ba2 larvae
[98]. (D–M) Immunohistochemical analyses of
fixed specimens. (D) Co-expression of mitfa::GFP (green) and sox10
(red). v, vertebral column. (E) mitfa::GFP+ cells aligned with
mbp+ glia (red) along ventral root motor fibers. Arrow,
mitfa::GFP+ cells did not co-express mbp. (F) Lateral view showing
mitfa::GFP+ cells and foxd3+ cells (red) between Hu+
neurons (blue) of dorsal root ganglia. mitfa::GFP+ and foxd3+
cells were often found close to one another (e.g., arrows) whereas other
cells co-expressed mitfa::GFP and foxd3 (arrowhead). (G) Lateral view
with superimposed brightfield and fluorescence images showing
mitfa::GFP+ and foxd3+ cells along a peripheral nerve fiber
stained for acetylated alpha tubulin (blue) within the myotome. Arrows,
adjacent mitfa::GFP+ and foxd3+ cells. (H). A nerve plexus
near the base of the caudal fin harbored numerous mitfa::GFP+ and
foxd3+ cells. (I) Along a peripheral nerve within the myotome some
cells co-expressed mitfa::GFP and foxd3 (arrowhead), whereas cells
expressing either mitfa::GFP+ or foxd3+ were often juxtaposed
(arrows). (J,K) Transverse sections through the dorsal trunk showing
sox10+ cells (J) and foxd3+ cells (K) n the hypodermis, within
the myotomes, and near the spinal cord. Arrow, lateral line nerve. (L,M)
In erbb3b mutant larvae, very few sox10+ (L) or
foxd3+ (M) cells were found.

The many extra-hypodermal mitfa::GFP+ cells in larvae contrasts with
embryogenesis [54] and suggests that extra-hypodermal cells may be
specified for the melanophore lineage during the larval-to-adult transformation.
If so, we predicted that cells expressing markers typical of early neural crest
cells (or glia) should occassionally be found to express mitfa::GFP as well. We
therefore examined larvae for simultaneous expression of mitfa::GFP, sox10, and
foxd3, and, to learn where these cells reside, we examined larvae with cell-type
specific markers for surrounding tissues. These analyses revealed numerous
mitfa::GFP+ cells associated with peripheral nerves and ganglia, including
the doral root ganglia, ventral motor root fibers, lateral line nerve, and nerve
fibers coursing through the myotomes (Figure 3E–3H; Figure S1).
Ectopic melanophores within the myotomes of wild-type
Tg(hsp70:kitla) larvae were likewise nerve-associated
(Figure
S2).

Double label analyses with markers of neural crest, glial, and melanophore
lineages revealed that, in wild-type larvae, mitfa::GFP+ cells were often
in close proximity to cells expressing sox10 and foxd3, and also co-expressed
sox10, as expected given the direct regulation of mitfa by
sox10 (Figure 3D, 3G, 3H)
[56],
[57]. We
also found that 4–15% of mitfa::GFP+ cells co-expressed foxd3,
with doubly labeled cells occurring most frequently during early metamorphosis,
when the rate of melanophore population increase is maximal [36] (Figure 3F, 3I; Figure 4A, 4B). This frequency
of double labeling is reminiscent of 15–18 h embryos, in which
9–12% of mitfa::GFP+ cells are foxd3+ [54]. In
contrast to the co-labeling of mitfa::GFP and foxd3, we did not find mitfa::GFP
expression by myelinating glia expressing myelin basic protein (mbp) (Figure 3E; Figure S1).
As anticipated, however, some cells expressing foxd3 or sox10 co-expressed mbp.
All of these cell types were deficient in erbb3b mutants and
wild-type larvae treated with AG1478 during the embryonic
erbb3b critical period (Figure 3J–3M; Figure 4A, 4B; Figure S4),
though a few residual mitfa::GFP+ and foxd3+ cells occurred in
anterior and posterior regions, corresponding to axial levels where a few
residual melanophores develop during metamorphosis [43] (data not shown).

If some foxd3+ and sox10+ cells are progenitors to post-embryonic
melanoblasts, we predicted that a period of population expansion could precede
the appearance of mitfa::GFP+ cells, which might themselves be
proliferative. Consistent with this idea, we found EdU incorporation by all
three cell types but EdU-labeling of post-embryonic foxd3+ and sox10+
cells was most frequent prior to pigment pattern metamorphosis, whereas
EdU-labeling of mitfa::GFP+ cells was most frequent during the peak of
pigment pattern metamorphosis (Figure 4C, Figure
5). Given these findings, we further asked if doubly labeled
foxd3+; mitfa::GFP+ cells constitute an especially proliferative
population. These analyses revealed EdU-incorporation in 55% of
foxd3+; mitfa::GFP+ cells, a significantly higher frequency than for
cells expressing only foxd3 (17% EdU+) or only mitfa::GFP
(24% EdU+;
χ2 = 131.7,
d.f. = 2, P<0.0001). The frequency of
EdU labeling amongst foxd3+; mitfa::GFP+ cells did not vary
significantly across stages
(χ2 = 2.3,
d.f. = 2, P = 0.3).
Finally, in erbb3b mutants sampled at selected stages, we found
lower levels of EdU incorporation than in wild-type, though small numbers of
cells overall resulted in correspondingly low statistical power (Figure 4C; Figure
S4).

All images from transverse sections of wild-type larvae staged as in
Figure 3 (see
Figure S4 for comparisons with erbb3b
mutant larvae). (A,B) Merged images showing cells (arrowheads) within
the myotomes labeled for either mitfa::GFP (red in A) or sox10::GFP (red
in B) as well as EdU (green). (C,D) Cells within the lateral myotomes
(m) and near the hypodermis (C) or at the base of the anal fin (f in D)
labeled for mitfa::GFP (red), foxd3 (blue), and EdU (green). Merged
views show fluorescence images or fluorescence images with brightfield
overlays. Arrowheads, triple-labeled cells. m, myotome. e,
epidermis.

The foregoing analyses indicated that, during post-embryonic development, a
proliferative population of extra-hypodermal, erbb3b-dependent
foxd3+ and sox10+ cells associated with peripheral nerves and ganglia
becomes specified as precursors to melanophores (or as bipotential precursors to
melanophores and iridophores; see reference [55]).

Extra-hypodermal cells contribute to the hypodermal populations of
metamorphic melanophores and iridophores

The development of post-embryonic, extra-hypodermal mitfa::GFP+ cells
suggested the possibility that such cells migrate to the hypodermis during
metamorphosis. To test this idea, we injected DiI into the myotomes, the
horizontal myoseptum, or the base of the dorsal fin of wild-type or
Tg(mitfa::GFP) larvae and we assessed after ≥4 d whether
DiI-labeled cells were present within the hypodermis distant from the injection
sites. In 10–30% of injected larvae we found hypodermal DiI-labeled
melanized cells, mitfa::GFP+ cells, or iridophores (Figure 6), indicating that some
extra-hypodermal cells migrated to the hypodermis during metamorphosis.

(A–C) DiI labeled tissues imaged immediately after injection into
the base of the dorsal fin (A), the vicinity of the horizontal myoseptum
and lateral line nerve (B), and the inner myotome (C). Each site yielded
hypodermal DiI+; mitfa::GFP+ cells or DiI+ melanophores
(12 of 30 larvae, 3 of 30 larvae, 15 of 87 larvae, respectively).
(D–F) DiI+ cells that expressed either mitfa::GFP (D) or
contained melanin (E,F) found within the lateral hypodermis 4 d
following injection into the base of the dorsal fin (D, F) or the inner
myotome (E). (G) DiI-labeling was observed for additional cells
including iridophores. Although the frequencies with which DiI labeled
pigment cells were found differed between injection sites, each site
gave rise to DiI+ iridophores at a frequency indistinguishable from
that of DiI+ mitfa::GFP+ cells
(χ2 = 0.6,
d.f. = 1,
P = 0.4). We did not observe
DiI-labeled xanthophores.

To further test the hypothesis that extra-hypodermal cells contribute to the
hypodermal melanophore population, we examined cell behaviors by time-lapse
imaging of trunks derived from Tg(mitfa::GFP) larvae. Movies
revealed the differentiation of mitfa::GFP+ cells into melanophores as well
as their migration (Figure
7A; Videos S4, S5, S6). We therefore assessed the migratory
pathways by which mitfa::GFP+ cells had reached the hypodermis.
Approximately half of all mitfa::EGFP+ cells arrived within the hypodermis
during imaging. Some entered the hypodermis after migrating over the dorsal or
ventral margins of the myotomes, whereas others originated from within the
myotomes, emerging either from the vicinity of the horizontal myoseptum or along
vertical myosepta (Figure
7B–7D, Figure
8; Videos S7, S8, S9, S10). Movies also revealed the movement of
mitfa::GFP+ cells along nerves and their detachment from nerves to migrate
more broadly through the fish (Videos S11, S12).
Together then, DiI labeling and time-lapse imaging indicated that
extra-hypodermal cells contribute to hypodermal mitfa::GFP+ cells,
melanophores, and iridophores during the larval-to-adult transformation.

All panels show lateral views of larval trunks and are derived from
time-lapse movies of Tg(mitfa::GFP) larvae. Elapsed
time (min) at lower right of each panel. (A) mitfa::GFP+ cells
differentiated into melanophores (e.g., arrowhead). (B–D)
mitfa::GFP+ cells entered the hypodermis during the larval-to-adult
transformation. (B) A cell at the dorsal margin of the myotomes extended
a long process (arrowhead) into the hypodermis and interacted with
processes of a second cell. *, cell body. (C) A long process
(arrowhead) preceded emergence of the cell body (*) from the level
of the horizontal myoseptum (dotted line). This cell subsequently
interacted with a neighboring cell, extended a processes ventrally, and
moved in that direction. (D) A cell initially deep within the myotome
(*) emerged into the hypodermis. The focal plane changes across
panels, from deep within the myotome to the surface of the myotome and
hypodermis, where other cells are found already. (E) Death of
mitfa::GFP+ cell (*) revealed by fragmentation and cellular
debris (arrowheads). (F,G) mitfa::GFP+ cells (F) and melanophores
that retain some residual GFP expression (G) proliferating within the
hypodermis. Melanophores in G are imaged in a kita
mutant (see text for details). See Videos
S4, S5, S6,
S7, S8, S9,
S10, S11, S12, S13, S14, S15, S16.

Shown are the relative frequencies of cells entering the hypodermis in
time-lapse movies of trunks derived from wild-type larvae
(N = 127 larvae, 1220
mitfa::GFP+ total cells examined) during different periods of
pigment pattern metamorphosis (very early, 6.4–6.6 SSL; early,
6.6–6.8 SSL; middle, 6.8–7.6 SSL). Cells newly arrived in
the hypodermis (n = 644) were
classified as migrating over the dorsal or ventral margins of the
myotomes (DV), emerging from the vicinity of the horizontal myoseptum
(HM) and lateral line, or emerging from within the myotomes (M),
tyically along vertical myosepta. Individuals were binned by stage and
bar widths are proportional to the average numbers of newly appearing
hypodermal mitfa::GFP+ cells per larva, normalized to cells
mm−2 day−1 (mean±SE above
bars). The relative frequencies of cells arising from different sources
differed between stages
(χ2 = 20.0,
d.f. = 4, P<0.0001), with cells
increasingly likely to emerge from within the myotomes as compared to
migrating over the dorsal or ventral margins of the myotomes.

Our findings suggested that a normal complement of adult melanophores depends on
contributions from a pool of extra-hypodermal precursors. If this is the case,
we predicted that mutants with severe metamorphic melanophore deficiencies
should have correspondingly severe deficiencies of extra-hypodermally derived
mitfa:GFP+ cells. To test the contributions of extra-hypodermal cells to
hypodermal mitfa::GFP+ cells and melanophores, we crossed the
Tg(mitfa::GFP) transgene into erbb3b and
tuba8l3a mutants, which exhibit severely reduced numbers of
metamorphic melanophores [36], [43], [44].

In comparison to the wild-type, and as predicted from the foregoing analyses,
erbb3b mutants had dramatically fewer extra-hypodermally
derived mitfa::GFP+ cells (Figure 9A; Video S13). erbb3b mutant
mifa::GFP+ cells originated from the vicinity of the horizontal myoseptum
(Figure 9B), and once in
the hypodermis, these cells were more likely to differentiate and to divide
(Figure 9C; in contrast
to the somewhat reduced rates of EdU incorporation prior to reaching the
hypodermis shown in Figure
4C). tuba8l3a mutants also had significantly fewer
extra-hypodermally derived mitfa::GFP+ cells. These cells were more likely
to differentiate, but divided at only one-third the frequency of the wild-type
(Figure 9; Video S14).
tuba8l3a mutants exhibit a post-embryonic demyelination of
the peripheral nervous system [44], and we found that regions exhibiting mbp+ glial
deficiencies and peripheral nerve defasciculation had fewer mitfa::GFP+ and
foxd3+ cells (Figure
10). We did not observe cells doubly labeled for foxd3 and mitfa::GFP in
tuba8l3a mutants.

Amongst the erbb3b- and tuba8l3a-dependent
metamorphic melanophore populations, are temporally and genetically distinct
subpopulations, comprising early metamorphic melanophores that are initially
dispersed but later migrate into stripes, and late metamorphic melanophores that
develop already at sites of stripe formation [37], [43], [45] (Figure S5).
Early metamorphic melanophores are ablated in kita mutants, but
persist in colony stimulating factor-1 receptor
(csf1r) and endothelin receptor b1
(ednrb1) mutants. By contrast, late metamorphic
melanophores persist in kita mutants, but are ablated in
csf1r and ednrb1 mutants [37], [38], [58], [59]. To test
if these differences reflect differential persistence of distinct precursor
pools, or differences in the subsequent morphogenesis and differentiation of
cells arising from a common precursor pool, we crossed the
Tg(mitfa::GFP) transgene into
kitab5, csf1rj4e1
and ednrb1b140 mutants and examined the origins of
hypodermal mitfa::GFP+ cells as well as their frequencies of
differentiation, death and proliferation.

In contrast to erbb3b and tuba8l3a mutants,
kita, csf1r, and ednrb1
mutants did not exhibit significantly fewer extra-hypodermally derived
mitfa::GFP+ cells than the wild-type, though cells in kita
mutants typically failed to differentiate and instead died at high frequency,
whereas cells in csf1r and ednrb1 mutants were
more likely both to differentiate and to die (Figure 9; Video S15).
We did not observe gross defects in mitfa:GFP+ cell motility in any of the
mutant backgrounds. Finally, in contrast to the proliferation of unmelanized
mitf::GFP+ cells (Figure
9C), proliferation of differentiated melanophores was rare in the
wild-type (0.1%; N = 3822
melanophores observed) and in most of the mutants (0.2–0.4%;
N = 4358 melanophore observed). In
kita mutants, however, the few melanophores that
differentiated divided frequently (14%;
N = 35 melanophores observed; variation
among genotypes:
χ2 = 38.1,
d.f. = 5, P<0.0001; Video S16).
Together these data show that erbb3b and
tuba8l3a each promote the development of extra-hypodermal
mitfa::GFP+ cells, whereas all five loci promote the differentiation and
morphogenesis of these cells once they reach the hypodermis.

Latent melanophore precursors in adult zebrafish

Our demonstration that extra-hypodermal precursors contribute to hypodermal
melanophores led us to ask whether latent pigment cell precursors persist into
the adult. Extra-hypodermal foxd3+ and mitfa::GFP+ cells were
distributed in adult fish similarly to metamorphic stages and also were found
associated with the scales (Figure S6). To test the capacity of latent
precursors to supply new melanophores, we sought to ablate melanophores with the
goal of provoking a regenerative response. Because fish doubly mutant for
kitab5 and presumptive null alleles of
csf1r lack body melanophores [38], we reasoned that fish
doubly mutant for kitab5 and the
temperature-sensitive allele
csf1rut.r1e174
(csf1rTS) [60] should have fewer
melanophores (equivalent to kitab5 single mutants)
at permissive temperature, but should lack all melanophores at restrictive
temperature. Repeated exposure to restrictive and permissive temperatures should
thus allow for repeated cycles of ablation and regeneration of these
kita-independent, csf1r-dependent
melanophores. As predicted, kita; csf1rTS double
mutants that were initially indistinguishable from kita single
mutants lost body melanophores when shifted to restrictive temperature (Figure 11A, 11B). After
returning to permissive temperature, fish initially recovered
kita-independent hypodermal melanophores, though
progressively fewer of these cells were regenerated in successive
ablation–recovery cycles (Figure 11C, 11D). Surprisingly, ablations also resulted in the
de novo development and regeneration of scale melanophores,
which are normally absent from kita mutants (Figure 11F, 11G)[37]. The few
later hypodermal melanophores that were recovered in kita;
csf1rTS mutants were often located beneath scales
populated with melanophores, iridophores and xanthophores (Figure 11H), raising the possibility that
some of these regenerative hypodermal melanophores may have been scale-derived.
Overall, these findings suggest that precursors to
kita-independent, csf1r-dependent hypodermal
melanophores persist in the adult yet have a finite regenerative potential,
whereas an additional precursor pool associated with adult scales has a greater
regenerative capability.

Discussion

The results of this study and previous analyses [36], [43], [45] suggest a model for the
development of adult melanophores in zebrafish (Figure 12). Pluripotent foxd3+ precursors to
glia [46], [61], adult
melanophores and iridophores are established in an erbb3b-dependent
manner during embryogenesis, and thereafter are associated with post-embryonic
peripheral nerves and ganglia. This precursor population is expanded and maintained
during pre-metamorphic larval development in an erbb3b- and
tuba8l3a-dependent manner, and cells within this pool become
specified for pigment cell lineages beginning immediately before, and continuing
through, pigment pattern metamorphosis. During the larval-to-adult transformation,
these extra-hypodermal precursors migrate to the hypodermis, and there contribute to
metamorphic melanophores and iridophores. Some enter the hypodermis after migrating
over the dorsal or ventral margins of the myotomes, others emerge from vertical or
horizontal myosepta; some may emigrate from the lateral line nerve. Once in the
hypodermis, these cells require tuba8l3a for their proliferation,
as well as kita, ednrb1, and, to a lesser extent,
csf1r, for their survival and eventual differentiation. Later
in adults, some latent precursors persist and can supply a limited number of new
hypodermal melanophores, whereas other precursors associated with scales have a
greater regenerative capacity. Below we discuss several aspects and implications of
this model.

Figure 12. Model for establishment and maintenance of adult pigment cell precursors
and their recruitment during development and regeneration.

(A) Hypothesized lineage relationships, showing neural crest (NC) cells in
the early embryo that give rise to Schwann cells and pigment cells of the
early larva as well as erbb3b-dependent progenitors to
metamorphic glial and pigment cell lineages (mGP). mGP are maintained in
association with peripheral nerves and ganglia, express foxd3, and their
population expands (multiple arrowheads) in a
tuba8l3a-dependent manner. During pigment pattern
metamorphosis (met), some mGP differentiate as metamorphic Schwann cells (S)
and the expansion of this lineage likely requires erbb3b
(not shown). Other mGP become specified for metamorphic pigment cell
lineages, as marked by mitfa::GFP expression. Some mitfa::GFP+ cells
will give rise to melanophores or iridophores and are initially
extra-hypodermally located in peripheral nerves and ganglia (M/I-e) but then
migrate to the hypodermis (M/I-h). The expansion of this population requires
ednrb1[58].
Some M/I-h will differentiate as metamorphic iridophores (I), other M/I-h
expand their population in a tuba8l3a- and
kita-dependent manner and ultimately differentiate as
metamorphic melanophores (M). Individual M/I-e or M/I-h may be bipotent for
melanophore and iridophore fates, as in embryos [55], or their respective
populations may harbor precursors already specified for either the
melanophore or iridophore fate. csf1r-dependent metamorphic
xanthophores (X) presumably arise from a different precursor population
(dashed line) and promote the survival of metamorphic melanophores (orange
arrow) [38], [42], [60]. Some
mGP persist into the adult and have a limited re-population potential. (B)
Schematic of metamorphic larva illustrating sources and migratory pathways
of metamorphic melanophore and iridophore precursors. Shown are mGP and
M/I-e (colors as in A) associated with nerves and beneath the dorsal fin
(f). M/I-e enter the hypodermis (arrows) from the dorsal or ventral margins
of the myotomes (m), or after migrating along nerves associated with the the
vertical myosepta (vm) or the horizontal myoseptum (hm). Others may arise
from the lateral line nerve (ll). Once in the hypodermis, these cells
differentiate as melanophores (green cell with heavy black outline) or
iridophores (not shown). sc, spinal cord. Additional populations of
precursors that may give rise to LM melanophores and scale melanophores are
not shown (see text).

Extra-hypodermal niches and the erbb3b- and
tuba8l3a-dependence of latent precursors to adult
melanophores

A major finding of our study is that post-embryonic mitfa::GFP+ cells are
associated with peripheral nerves coursing through the myotomes as well as more
medial nerves and ganglia. We further showed that nerve-associated cells could
be induced to differentiate ectopically as melanophores, and that
extra-hypodermal mitfa::GFP+ cells migrate to the hypodermis where some
differentiate as melanophores during normal development. These observations
suggest that peripheral nerves or ganglia serve as niches for post-embryonic
precursors to adult melanophores and are broadly consistent with a recent study
demonstrating a peripheral nerve origin for adult skin melanocytes of amniotes
[62] as
well as analyses revealing interconversion of glial and melanocyte fates
in vitro[63]–[65]. Our study complements and extends recent lineage
tracing studies of flounder larvae, in which adult pigment cell precursors were
found to migrate to the hypodermis from dorsal and ventral regions during
pigment pattern metamorphosis [66], [67].

Our analyses also provide insights into the molecular and proliferative
phenotypes of metamorphic melanophore precursors. foxd3 often acts as a
transcriptional repressor and is associated with the maintenance of pluripotency
and pluripotent cells [68]–[71]. In the neural crest
lineage, foxd3 is expressed by pluripotent cells and presumptive glia, and can
inhibit mitfa transcription, favoring iridophore or glial over
melanogenic fates [54], [55], [61], [72]. We found that some nerve-associated foxd3+
cells co-expressed mitfa::GFP just before and continuing through pigment pattern
metamorphosis, and that such cells were especially likely to have incorporated
EdU. These observations raise the possibility that nerve-associated foxd3+
cells are a pluripotent and proliferative population that can give rise to
hypodermal melanophores and iridophores during pigment pattern metamorphosis.
Our finding that DiI-labeled, extra-hypodermal tissues give rise to melanophores
and iridophores with equal frequency is consistent with this idea. Because the
mitfa::GFP transgene we employed is repressible by foxd3, we speculate that
co-expression of mitfa::GFP reflects low levels of perduring foxd3 protein as
precursors adopt a pigmentary fate, similar to observations of early neural
crest morphogenesis [54], or that a balance between the anti-melanogenic and
pro-melanogenic effects of foxd3 and mitfa, respectively, prevents specified
cells from differentiating prematurely. Nevertheless, we note that our analyses
revealed more extra-hypodermal mitfa::GFP+ cells than
mitfa+ cells, which differs from the one-to-one
corrrespondence of such cells in embryos [54]. Additional
mitfa::GFP+ cells could reflect low levels of mitfa
expression that fall below the threshold for detection by in situ hybridization
at post-embryonic stages, yet are sufficient for accumulating detectable levels
of relatively stable GFP. Or, mitfa::GFP expression in some cells could reflect
a partial disregulation of the transgene, as might occur if regulatory elements
for post-embryonic expression are missing. Distinguishing between these
possibilities will require the production and analysis of additional transgenic
reporter lines, but whichever the outcome, the mitfa::GFP reporter we have used
will be a valuable tool for further dissecting the mechanisms of post-embryonic
melanophore development.

Examination of erbb3b and tuba8l3a mutants
provides additional support for the idea that extra-hypodermal, nerve-associated
precursors are essential for metamorphic melanophore development. We found that
presumptive precursors to glia and pigment cells were missing from
erbb3b mutants and wild-type larvae in which ErbB activity
had been inhibited during the embryonic critical period for adult melanophore
development. An on-going requirement for erbb3b is suggested as
well, by reduced complements of adult melanophores following acute inhibition of
ErbB signaling in sensitized backgrounds during pigment pattern metamorphosis
[43], and by
reduced rates of EdU incorporation in erbb3b mutants during
post-embryonic development (this study). Because ErbB signaling can repress
melanocyte differentiation [62], [73], a further role in preventing the premature
differentiation of nerve-associated precursors to hypodermal pigment cells seems
likely. Notably, our finding that extra-hypodermal, kitla-responsive melanogenic
cells were missing in ErbB-deficient backgrounds at post-embryonic stages
contrasts with increased numbers of such cells at embryonic/early larval stages
[74].
This difference likely reflects the pleiotropic nature of ErbB signals and a
difference between the stages examined: blocking ErbB activity in the embryo
presumably results in a de-repression of melanophore differentiation amongst
transiently persisting precursor cells, whereas by post-embryonic stages, such
precursors presumably have been lost.

In contrast to erbb3b mutants, tuba8l3a mutants
exhibit a post-embryonic demyelination of peripheral nerves and a corresponding
critical period for adult melanophore development [36], [44], [45]. In agreement with our
model, tuba8l3a mutant larvae were deficient for mbp+
glia, nerve-associated foxd3+ cells and foxd3+; mitfa::GFP+
cells, and also had reduced rates of division amongst hypodermal
mitfa::GFP+ cells. Because tuba8l3a acts autonomously to
the metamorphic melanophore lineage [45], these findings suggest a
defect in the maintenance or expansion of pluripotent precursors to mbp+
glia and mitfa:GFP+ pigment cell precursors. The post-embryonic onset of
these phenotypes further suggests the existence of genetically distinct
populations of embryonic and adult glia, analogous to embryonic and metamorphic
melanophores.

Time-lapse imaging comparisons of wild-type and mutant backgrounds further
defined roles for genes previously known to function in pigment pattern
development: kita, csf1r, and
ednrb1 all promoted the survival of mitfa::GFP+ cells
whereas kita also promoted the differentiation of these cells
as melanophores. Yet, these analyses revealed compensatory responses of pigment
cells and their precursors in mutant backgrounds as well. For example, residual
mitfa::GFP+ cells in mutants with extra-hypodermal precursor deficiencies
(erbb3b, tuba8l3a) or hypodermal defects
in cell survival (csf1r, ednrb1) exhibited
concomitantly greater rates of differentiation, and, in one instance
(erbb3b), an increased rate of proliferation. Moreover, the
reduced survival and differentiation of mitfa::GFP+ cells in
kita mutants was coupled with a 70-fold increase in
melanophore proliferation. These findings highlight the remarkably regulative
nature of zebrafish pigment pattern development [36], [41], [42] and also the importance
of direct imaging for understanding cellular behaviors that would not be
predicted from terminal phenotypes alone.

A particularly dramatic example of pattern regulation occurs during regeneration.
Zebrafish larval melanophores regenerate following laser ablation or the
administration of melanocytotoxic drugs [74]–[76], adult fin
melanophores regenerate along with other tissues after fin amputation [77]–[79], and
hypodermal body melanophores regenerate after localized laser ablations [80], [81]. To test
the capacity of latent precursors to supply new hypodermal melanophores in the
adult, we used fish doubly mutant for kita and
csf1rTS in which loss of residual
melanophores at restrictive temperature likely reflects the withdrawal of
trophic support provided by csf1r-dependent xanthophores [42], [60]. Our
finding that progressively fewer hypodermal melanophores were recovered after
repeated ablations implies a limited re-population potential for latent
precursors that give rise to these kita-independent,
csf1r-dependent late metamorphic melanophores. Whether the
same is true of kita-dependent early metamorphic melanophores
remains to be determined. Nevertheless, our finding that scale melanophores
regenerated repeatedly-in contrast to late metamorphic, hypodermal
melanophores-suggests a more highly regulative precursor pool associated with
the adult scales, and highlights the possibility of spatially and temporally
distinct pools of precursors having different morphogenetic and differentiative
potentials. That scale melanophores developed de novo in these
fish, despite their absence from kita single mutants, likely
reflects a priority effect; e.g., initially abundant xanthophores may repress
melanophore development in kita mutants [42], [82], [83] but simultaneous
development of both melanophores and xanthophores during regeneration allows for
a stable pattern comprising both cell types. The similar re-population potential
of scale and fin melanophores, and the previous observation that
basonuclin-2 mutants, which are deficient for hypodermal
melanophores, nevertheless retain both scale and fin melanophores [84], also suggest
the possibility of more extensive similarities between scale- and fin-associated
precursor pools.

Finally our study provides new insights into the temporally distinct populations
of melanophores in zebrafish. We found severe deficiencies in the numbers of
extra-hypodermally derived mitfa::GFP+ cells in erbb3b and
tuba8l3a mutants, illustrating the critical role of such
cells in supplying metamorphic melanophores overall. By contrast, we found no
evidence for reduced numbers of extra-hypodermally derived mitfa::GFP+
cells in kita, csf1r, or
ednrb1 mutants, indicating that different genetic
requirements of early and late metamorphic melanophores do not reflect
differences in the establishment or maintenance of these cells. These findings
suggest either of two interpretations. Early and late metamorphic melanophores
could arise from a common precursor pool with differences in residual
melanophore complements among mutants reflecting specific requirements for
kita, csf1r, and ednrb1
in downstream events of morphogenesis and differentiation. For example, our data
indicate that mitfa::GFP+ cells require kita for their
surival, and presumably, terminal differentiation: the development of late
metamorphic melanophores in kita mutants could thus reflect the
late appearance of factors able to substitute for kita activity
in promoting melanophore differentiation. Conversely, we found that
mitfa::GFP+ cells were only marginally dependent on csf1r:
the failure of late metamorphic melanophores to develop in
csf1r mutants could, in turn, reflect a late-arising,
post-differentiation requirement for trophic support from
csf1r-dependent xanthophores [38], [42], [60]. An alternative
interpretation is that early and late metamorphic melanophores do arise from
distinct, but still cryptic, precursor pools that simply were not revealed by
our methods. In other systems, niches initially assumed to have just one type of
stem or progenitor cell have sometimes been found to harbor distinct classes of
cells with disparate proliferative or differentiative potentials [85]–[87]. Additional
time-lapse imaging analyses at later stages and genetically based lineage
analyses that are now being conducted should provide further insights into these
possibilities.

Post-embryonic stem cells in development, evolution, and neoplasia

The identification of extra-hypodermal nerve-associated precursors to adult
melanophores in zebrafish (this study) and amniotes [62] indicates that a fuller
understanding of adult pigment cell development and pattern formation requires a
focus on post-embryonic precursors, as distinct from embryonic neural crest
cells. The existence of genetically separable populations of embryonic pigment
cells, derived directly from neural crest cells, and adult pigment cells,
derived from post-embryonic precursors, further suggests that
species-differences in adult pigment patterns may be explicable by evolutionary
changes in the establishment, maintenance or recruitment of post-embryonic
latent precursors, with few if any consequences for earlier pigment patterns
[88].
Lastly, a peripheral nerve origin for adult pigment cells also raises the
possibility that the frequent and generally fatal metastases of melanoma cells
to the central nervous system [26]–[29],[89],[90] may reflect the continued or reiterated expression of
genes that favor proliferation and migration in a nerve microenvironment.

Materials and Methods

Fish rearing, staging, and genetic stocks

Fish were reared at 28–29°C, 14L:10D. Post-embryonic stages are
reported as standardized standard length (SSL) measurements, which indicate
developmental progress of free-feeding larvae more reliably than days
post-fertilization [33]. Tg(mitfa::GFP)w47 and
Tg(−4.9sox10:egfp)ba2 fish were
generously provided by D. Raible and R. Kelsh, respectively.
Tg(TrDct::mCherry)wp.r.t3 and
Tg(hsp70::kitla)wp.r.t2 fish were produced
using tol2kit Gateway vectors and Tol2-mediated transgenesis
[91],
[92] and
heat shocks with the latter strain were administered at 37°C for 1 hr three
times daily for two days. Experiments with erbb3b mutants used
either of two presumptive null alleles,
erbb3but.r2e1 or
erbb3bwp.r2e2[93].
Experiments with csf1r mutants used either the presumptive null
allele csf1rj4blue or the temperature-sensitive
allele csf1rut.r1e174[60]. In
temperature shift experiments, fish were shifted repeatedly between restrictive
temperature (33°C) and permissive temperature (24°C). All experiments
with the kita mutant used the presumptive null allele
kitab5[59].
tuba8l3aj115e1 encodes a missense
substitution with temperature-sensitive effects. Experiments with this allele
were performed at standard rearing temperature, intermediate between restrictive
(33°C) and permissive (24°C) temperatures [36], [44], [45], to allow analyses of a
fuller complement of mitfa::GFP+ cells. Quantitative analyses here are thus
likely to underestimate effects of the tuba8l3a mutation.
Animal use conformed to University of Washington IACUC guidelines.

Imaging and image analysis

Fish were viewed with Olympus SZX-12 or Zeiss Discovery epifluorescence
stereomicroscopes or with a Zeiss Observer inverted compound epifluorescence
microscope with Apotome. Images were collected in Axiovision software using
Axiocam HR or MR3 cameras. For thick specimens, stacks of images were collected
and processed using Zeiss Axiovision 6D Acquisition or Extended Focus modules
and some fluorescence images were deconvolved using the Zeiss Axiovision
Deconvolution module. Alternatively, specimens were viewed and images collected
on Zeiss 510 META or Olympus FV1000 laser confocal microscopes.

DiI injection

Cell Tracker CM-DiI (Invitrogen) was prepared as a stock solution in DMSO then
diluted to 0.025–0.05% in 0.3 M sucrose just before use. Larvae
were anesthetized briefly and injected with 1–2 nl of DiI using a
borosilicate needle, imaged immediately to ascertain the specificity of staining
in target tissues, then reared individually until analyzed.

Ex vivo time-lapse imaging

Larvae were rinsed with 10% Hanks medium, anesthetized and then sacrificed
by decapitation using a razor blade. After removing the anterior portion of the
trunk and discarding the tails, larval trunks were placed on 0.4 µm
transwell membranes (Millipore) in glass-bottom dishes containing L-15 medium,
3% fetal bovine serum, and penicillin/streptomycin. Trunks were
equilibrated at 28.5°C for 3 h then imaged for 18–24 h (20 or 30
minute intervals between images) on a Zeiss Observer inverted epifluorescence
microscope. Comparisons between isolated trunks imaged continuously for 24 h and
repeatedly anesthetized intact larvae did not reveal gross differences in the
survival of mitfa::GFP+ cells, though average maximal estimated velocities
of mitfa::GFP+ cells were reduced by ∼22% in cultured trunks as
compared to intact larvae (P<0.05;
N = 67 cells examined). Imaging over
longer durations resulted in increased rates of cell death throughout the
explant and thus were not used for analyses.

Pharmacological inhibition of ErbB activity

A stock solution of AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline;
Calbiochem] was diluted in DMSO. Embryos were treated with 3 µM
AG1478 in 10% Hanks for through either 72 or 96 hours post-fertilization.
To facilitate penetration, 0.5% DMSO was added to all media and embryos
were dechorionated prior to treatment. Fish were reared in glass Petri dishes
and solutions were changed daily.

EdU labeling

Larvae were incubated with 0.005% 5-ethynyl-2′-deoxyuridine (EdU;
Intvitrogen) in 10% Hank's medium containing 1% DMSO for
36 h. Larvae were then sacrificed, fixed with 4% PFA/1% DMSO, and
vibrotome sectioned (150–200 µm) for immunohistochemistry followed
by histochemical detection of EdU according to manufacturer's
recommendations.

Statistical analyses

Quantitative data were analyzed with JMP 8.0.2 (SAS Institute, Cary NC).
Frequency data were examined using multiple logistic regression or contingency
table analyses, and tested for effects of genotype, stage, and genotype x stage
interactions. Significance of effects were assessed by likelihood ratio tests
and non-significant factors were removed from the final models. Analyses of
variance were used for continuous variables including counts. Residuals were
examined for normality and homogeneity of variances, conditions that were
achieved for some variables after transformation by square root or natural
logarithm. Further details of statistical analyses are available upon
request.

Supporting Information

mitfa::GFP+ cells amongst glia of the lateral line nerve. Sagittal view
of a wild-type larva showing mitfa::GFP+ cells (mitfa, green; arrow)
aligned with mbp+ glia (red) of the main trunk lateral line nerve, near
the horizontal myoseptum.

Deficiencies in mitfa::GFP+ cells, foxd3+ cells, and EdU
incorporation in erbb3b mutants. (A,B) Views and
annotations correspond to those for wild-type larvae in main text Figure 5. Arrow, a rare
EdU+; mitfa::GFP+ cell at the base of the ventral fin.

mitfa::GFP+ cells in a living wild-type larva. Images were collected in
sagittal planes from the skin to the midline of the trunk. Image rotation
reveals extra-hypodermal cells within the myotomes, that typically expressed
mitfa::GFP at levels lower than in hypodermal cells.

mitfa::GFP+ and dct::mCherry+ melanophores in a living wild-type
larva. In contrast to mitfa::GFP+ cells (green), cells expressing the
later melanophore lineage marker dct were detected only in
the hypodermis. Shown here are cells expressing mCherry driven by the
Takifugu rubripes dct promoter.

mitfa::GFP+ cells are missing from a live erbb3b mutant
larva. Larva imaged as in Video S1 but over-exposed to provide
spatial context. Fluorescent cells forming a stripe in the hypodermis are
iridophores, which reflect under epifluorescence in multiple channels (these
cells are present in the larva shown in Video
S1, but their reflectance falls beneath the threshold for
detection at the exposure shown).

:GFP+ cells. Low magnification view showing highly migratory
mitfa::GFP+ cells over ∼18 h. This movie loops three times.
mitfa::GFP+ cells are highly migratory and extend long probing
processes. These cells can be seen migrating widely over the flank and also
migrating from the body into the anal fin at the lower edge of the frame. In
contrast to mitfa::GFP+ cells, xanthophores are larger, less motile and
autofluoresce weakly in the GFP channel, particularly from accumulations of
pteridine-containing organelles around the nucleus.

mitfa::GFP+ cells emerging from within the myotome. High magnification
view showing a single mitfa::GFP+ cell (arrow) emerging from within the
myotome. In this movie, only selected planes of focus from a complete
z-series are shown, illustrating the progressive movement of this cell from
the interior myotome to the surface. As the cell approaches the more
superficial planes, additional mitfa::GFP+ cells already in the
hypodermis come into focus.

mitfa::GFP+ cells entering the hypodermis after migrating over the
dorsal myotome. One cell already at the dorsal myotome and another extending
a process over the flank at the beginning of the movie (red arrows). A third
cell extends a long filopodium (yellow arrow) over the flank before entering
into the hypodermis.

mitfa::GFP+ cells migrating within the caudal nerve plexus. Shown are
mitfa::GFP+ cells along a nerve plexus near the base of the caudal fin
in the medial region of a larva. This particular individual is a
kita mutant. Similar behaviors were observed in
wild-type larvae.

Proliferation of differentiated melanophores. Shown is the posterior tail of
a kita mutant larva, illustrating successive melanophore divisions.
mitfa::GFP+ cells also can be observed migrating on the body and
between the body and caudal fin.

doi:10.1371/journal.pgen.1002044.s022

(MOV)

Acknowledgments

Thanks to Dave Raible, Kevin Curran, and other members of the Raible lab for
providing reagents and for helpful discussions; members of the Parichy lab for
assistance with fish rearing; and David Lyons, Will Talbot, Bruce Appel, and Robert
Kelsh, who generously provided reagents.

Author Contributions

Conceived and designed the experiments: EHB LBP DMP. Performed the
experiments: EHB LBP DMP. Analyzed the data: EHB DMP. Wrote the paper: EHB
LBP DMP.

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